Autoimmune diseases and transplantation related problems remain major public health problems worldwide. Although immunosuppressive drugs are in wide use and have shown effectiveness, their clinical usefulness has been limited due to their toxicity and other short- and long-term side effects such as risk of malignancy. Currently there is a need for new immunomodulatory agents with improved immunosuppressant activity and pharmacokinetic properties, improved bioavailability, greater potency, extended effective half-life in vivo, fewer side effects and less complicated dosing schedules.
Key to immune function and transplantation rejection are the major histocompatibility antigens, commonly referred to in humans as the HLA complex. Genes encoding class I HLA proteins are clustered at the telomeric end of human chromosome 6p21. These include the classical class Ia proteins, HLA-A, -B and -C, which are ubiquitously expressed, and are highly polymorphic. In contrast, the non-classical class Ib proteins, HLA-E, HLA-F and HLA-G, are relatively invariant, and are selectively expressed.
HLA-G is an antigen of great interest, and the focus of significant experimental evaluation. Features important to the function of this molecule include a low number of functionally different alleles; seven alternatively spliced transcripts, four encoding membrane-bound proteins and three encoding soluble proteins; and the generation of proteins with a truncated cytoplasmic tail, revealing a cryptic retrieval motif that may interfere with presentation of exogenous peptides.
In HLA-G, nine polymorphisms have been identified in the exons encoding the extracellular domain, and one in the 3′ untranslated region. Of the nine, five result in amino acid differences and four do not. One major deletion has been identified: a single base pair (bp) deletion at nucleotide 1597, which causes a frameshift at amino acid 130. This deletion of a cytosine residue at codon 130 results in a null allele (called G*0105N), which does not encode functional HLA-G1 or HLA-G5 protein isoforms.
The full-length isoform, HLA-G1, is structurally similar to other class I genes, except for the truncated cytoplasmic tail. The G2 isoform results from the removal of exon 3; the resulting heavy chain cannot form heterodimers with β2 microglobulin and homodimerizes to form an HLA class-II-like structure. HLA-G1 and HLA-G2 are also expressed as soluble proteins (called HLA-G5 and HLA-G6, respectively) because of the inclusion of intron 4 sequences in the mature mRNA. HLA-G5 and HLA-G6 secreted proteins include a unique sequence of 21 amino acids. HLA-G5 may or may not associate with β2m, whereas HLA-G6 does not. HLA-G3 results from the removal of exons 3 and 4. HLA-G4 and HLA-G7 mRNAs are scarce in placentas, and the functions of their protein products remain unknown. The soluble HLA-G isoforms circulate in mothers' blood throughout pregnancy. Soluble HLA-G is also produced by some but not by all preimplantation embryos.
HLA-G is involved in the induction of immune tolerance. Its effects include impact on NK cell killing, migration, and cell viability; proliferation and IFNγ production; regulation of cytokine production in blood mononuclear cells and cytotoxic T lymphocytes (CTLs); suppression of CTL killing and viability; inhibition of proliferation and induction of a suppressive phenotype in T-helper cells; and alteration of dendritic cell stimulatory capacity and maturation of this lineage.
The major receptors for HLA-G on leukocytes are the leukocyte-inhibitory receptors (LILRB), formerly known as the immunoglobulin-like transcript (ILT) receptors. LILRBs are expressed by T and B lymphocytes and also by NK cells and mononuclear phagocytes, and LILRBs abrogate activating signals received by these cells. Although LILRB1 (ILT2) appears to be the main binding protein for lymphocytes, LILRB2 (ILT4) may be the main receptor for HLA-G, which is exhibited by monocyte/macrophages, the second most populous leukocyte population in the human decidua.
Similar to HLA-G, cell surface expression of HLA-E is limited, although it is transcribed in all human tissue. The limited number of peptides capable of binding to HLA-E include nonamer peptides derived from the signal sequence of classical MHC or of HLA-G molecules and stress-associated autologous and pathogen molecules. When bound to signal peptides from classical MHC class I molecules, HLA-G or CMV, HLA-E triggers the inhibitory NK receptor, CD94/NKG2A.
Since most NK cells and T cells express CD94/NKG2A, it is hypothesized that expression of HLA-E inhibits the activities of CD94/NKG2A-expressing effector cells. Indeed, induction of HLA-E expression by target cells led to a significant inhibition of both lysis and cytokine secretion by CD94/NKG2A-expressing NK cells or CTL (see, e.g., Borrego et al. (1998) J. Exp. Med. 187: 813-818; Braud et al (2003) Trends Immunol. 24: 162-164; Le Drean et al. (1998) Eur. J. Immunol. 28: 264-276).
The therapeutic implication of cells expressing HLA-G and/or HLA-E is tremendous. Identification of long-lived stem cell populations that express these proteins are of interest for various clinical and research purposes. The present invention addresses this need.